PROJECT SUMMARY/ABSTRACT WNT signaling is crucial for embryonic development and adult tissue homeostasis, with aberrant signaling resulting in developmental disorders and disease, including cancer. Although much is known, a deeper understanding of this signaling cascade will improve our understanding of cancer genesis and progression, and will empower the development of effective therapeutics. WNT/b-catenin signaling is driven by the stabilization of the transcriptional co-activator, b-catenin. In the absence of WNT ligand, a cytosolic destruction complex phosphorylates, ubiquitylates and degrades of b-catenin. In the presence of WNT ligand, an alternative complex called the LRP6 signalosome forms on the intracellular side of the plasma membrane, in association with the WNT receptors, LRP6 and Frizzled. This results in b-catenin accumulation and activation of b-catenin target genes. Recent data demonstrate that upon WNT ligand engagement, the signalosome is endocytosed. Although conflicting data exist within the literature, a consensus is beginning to emerge that clathrin-dependent endocytosis of the signalosome results in its degradation. This training proposal and my thesis project is devoted to elucidating the molecular events and dynamics of signalosome endocytosis in normal cells and in cancer. To this end, we used a gain-of-function screen of the kinome to identify AAK1, a regulator of clathrin-mediated endocytosis as a negative regulator of WNT signaling. My preliminary data suggests AAK1 decreases surface levels of LRP6. I will further evaluate the role of AAK1 in regulating signalosome endocytosis in multiple cancer cell models. I will also define the AAK1 protein-protein interaction network and its WNT3A-dependent dynamics by quantitative mass spectrometry. Specifically, proteomics analysis of the LRP6 and AAK1 interaction networks in response to an extended Wnt3A time course will provide insight into the dynamic changes to signalosome composition and potentially identify new regulators of WNT signaling. My lab previously discovered the WTX tumor suppressor as a component of the signalosome and b-catenin destruction complex. My preliminary data demonstrates that WTX interacts with AAK1 and other regulators of clathrin-mediated endocytosis. Therefore, my training proposal also aims to describe a role for WTX in promoting signalosome endocytosis via AAK1. I will define a comprehensive WTX protein-protein interaction network by quantitative mass spectrometry, again at varying times post WNT3A treatment. To summarize, the precise role of endocytosis in WNT signaling remains unclear, with numerous questions surrounding the mechanism(s) and components of endocytosis and its effects on signaling. Therefore, the central hypothesis for my proposal is that following WNT stimulation, AAK1 and WTX cooperatively promote clathrin-mediated endocytosis of LRP6 and the signalosome. This work will provide me training in and experience in the mechanisms of WNT signaling and feedback attenuation.